Japanese encephalitis virus (JEV) is the most common cause of viral encephalitis in Asia and parts of the Western Pacific, with ˜60% of the world's population at risk of infection. Within the family Flaviviridae (genus Flavivirus), JEV belongs to the JE serological group, which also includes medically important human pathogens found on every continent except Antarctica: West Nile virus (WNV), St. Louis encephalitis virus (SLEV), and Murray Valley encephalitis virus (MVEV). Historically, the JE serological group members have clustered in geographically distinct locations, but the recent emergence and spread of JEV in Australia and WNV in North America have caused growing concern that these viruses can spread into new territory, posing a significant challenge for global public health. In the US, where WNV and SLEV are endemic, the situation is particularly problematic because the likelihood of JEV being introduced is considerable. Worldwide, ˜50,000-175,000 clinical cases of JE are estimated to occur annually; however, this incidence is undoubtedly a considerable underestimate because surveillance and reporting are inadequate in most endemic areas, and only ˜0.1-4% of JEV-infected people develop clinical disease. On average, ˜20-30% of patients die, and ˜30-50% of survivors suffer from irreversible neurological and/or psychiatric sequelae. Most clinical cases occur in children under age 15 in endemic areas, but in newly invaded areas, all age groups are affected because protective immunity is absent. Thus, given the current disease burden and significant threat of the JEV emergence, resurgence, and spread among much larger groups of susceptible populations, control of JEV remains a public health priority.
JEV contains a nucleocapsid composed of an ˜11-kb plus-strand genomic RNA, complexed with multiple copies of the highly-basic α-helical C proteins. The nucleocapsid is surrounded by a host-derived lipid bilayer containing the membrane-anchored M and E proteins. The initial step in the flavivirus replication cycle involves attachment of the virions to the surface of susceptible cells. The viral E protein is then assumed to bind with high affinity and specificity to an as-yet unidentified cellular receptor(s), which triggers receptor-mediated, clathrin-dependent endocytosis. The acidic conditions in the endosome lead to a conformational change in the E protein, which triggers fusion of the viral membrane with host endosomal membrane. Once the genome is released into the cytoplasm, the genomic RNA is translated into a single polyprotein, which is processed co- and post-translationally by host and viral proteases to yield at least 10 functional proteins: three structural (C, prM, and E) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The nonstructural proteins actively replicate the viral genomic RNA in the replication complex that is associated with the virus-induced, endoplasmic reticulum (ER)-derived membranes. Newly synthesized genomic RNA and C proteins are initially enveloped by the prM and E proteins to generate immature virions that bud into the lumen of the ER. These immature virions are then transported via the secretory pathway to the Golgi apparatus. In the low-pH environment of the trans-Golgi network, the furin-mediated cleavage of prM to M induces the maturation of the viral particles, which is also accompanied by significant structural rearrangements of the M and E proteins. Finally, mature virions are released into the extracellular space by exocytosis.
JEV is maintained in an enzootic cycle involving multiple species of mosquito vectors (primarily Culex species) and vertebrate hosts/reservoirs (mainly domestic pigs/wading birds). Humans become infected incidentally when bitten by an infected mosquito. In the absence of antiviral therapy, active immunization is the only strategy for sustainable long-term protection. Four types of JE vaccines are used in different parts of the world: (i) the mouse brain-derived inactivated vaccine based on the Nakayama or Beijing-1 strain, (ii) the cell culture-derived inactivated vaccine based on the Beijing-3 or SA14-14-2 strain, (iii) the cell culture-derived live-attenuated vaccine based on the SA14-14-2 strain, and (iv) the live chimeric vaccine developed on a yellow fever virus (YFV) 17D genetic background that carries two surface proteins of JEV SA14-14-2. Of the four vaccines, the only one that is available internationally is the mouse brain-derived inactivated Nakayama. Unfortunately, the production of this vaccine was discontinued in 2006 because of vaccine-related adverse events, short-term immunity, and high production cost. To date, the most commonly used vaccine in Asia is the live-attenuated SA14-14-2, but this vaccine is not recommended by the World Health Organization for global immunization. In addition to the duration of immunity in relation to the number of doses, the most critical issue with this vaccine remains a risk for reversion of the virus to high virulence. Recently, the SA14-14-2 vaccine virus has been utilized to produce a new Vero cell-derived inactivated vaccine that has been approved in the US, Europe, Canada, and Australia since 2009. In the US, this vaccine is recommended for adults aged 17 years travelling to JEV-endemic countries and at risk of JEV exposure, but no vaccine is currently available for children under 17. More recently, the prM and E genes of JEV SA14-14-2 have been used to replace the corresponding genes of YFV 17D, creating a live chimeric vaccine that is now licensed in Australia and Thailand. Thus, the application of JEV SA14-14-2 to vaccine development and production is continuously expanding, but the viral factors and fundamental mechanisms responsible for its loss of virulence are still elusive.
The virulence of JEV is defined by two properties: (i) neuroinvasiveness, the ability of the virus to enter the central nervous system (CNS) when inoculated by a peripheral route; and (ii) neurovirulence, the ability of the virus to replicate and cause damage within the CNS when inoculated directly into the brain of a host. Over the past 20 years, many investigators have sought to understand the molecular basis of JEV virulence, by using cell and animal infection model systems to compare the nucleotide sequences of the genomes of several JEV strains that differ in virological properties. These studies have identified a large number of mutations scattered essentially throughout the entire viral genome. Because of the complexity of the mutations, however, the major genetic determinant(s) critical for either JEV neurovirulence or neuroinvasiveness remains unclear. In particular, the situation is more complicated for the live-attenuated SA14-14-2 virus, which has been reported to have a number of different mutations, i.e., 47-64 nucleotide changes (17-27 amino acid substitutions), when compared to its virulent parental strain SA14; the exact number depends on both the passage history of the viruses and the type of cell substrate used for virus cultivation. A more comprehensive sequence comparison with another SA14-derived attenuated vaccine strain, SA14-2-8, together with two other virulent strains, has suggested seven common amino acid substitutions that may be involved in virus attenuation: 4 in E, 1 in NS2B, 1 in NS3, and 1 in NS4B. However, the genetic component directly responsible for the attenuation of SA14-14-2 is still unknown. Given that SA14-14-2 has been administered to >300 million children for >20 years in China and recently in other Asian countries, it is striking that there is a fundamental gap in our knowledge at the molecular level about how SA14-14-2 is attenuated.